Live-Cell Imaging of Cytoskeletal Responses and Trafficking During Fungal Elicitation.

Understanding the mechanisms driving plant defense responses holds the promise to provide new means to reinforce plant defense both through agrochemicals and targeted genetic improvement. The capability to quantify impacts of phytopathogens on subcellular dynamics is particularly important when elucidating the role of specific virulence mechanisms that make contributions toward infection success but do not individually alter disease outcome. Acquiring these data requires an investigator to achieve the successful handling of both plant and microbe prior to observation and an appreciation of the challenges in acquiring images under these conditions. In this chapter we describe a protocol to support the observation of cytoskeletal dynamics surrounding sites of fungal interaction, specifically the powdery mildew Blumeria graminis f.sp. hordei on the surface of Arabidopsis thaliana. Furthermore, we also describe a procedure to expose etiolated (dark-grown) hypocotyls to a molecular pattern to activate defense responses in the absence of a phytopathogen with the aim of observing localized actin-dependent trafficking.

An Immune-Responsive Cytoskeletal-Plasma Membrane Feedback Loop in Plants.

Cell wall appositions (CWAs) are produced reactively by the plant immune system to arrest microbial invasion through the local inversion of plant cell growth. This process requires the controlled invagination of the plasma membrane (PM) in coordination with the export of barrier material to the volume between the plant PM and cell wall. Plant actin dynamics are essential to this response, but it remains unclear how exocytosis and the cytoskeleton are linked in space and time to form functional CWAs. Here, we show that actin-dependent trafficking to immune response sites of Arabidopsis thaliana delivers membrane-integrated FORMIN4, which in turn contributes to local cytoskeletal dynamics. Total internal reflection fluorescence (TIRF) microscopy combined with controlled induction of FORMIN4-GFP expression reveals a dynamic population of vesicular bodies that accumulate to form clusters at the PM through an actin-dependent process. Deactivation of FORMIN4 and its close homologs partially compromises subsequent defense and alters filamentous actin (F-actin) distribution at mature CWAs. The localization of FORMIN4 is stable and segregated from the dynamic traffic of the endosomal network. Moreover, the tessellation of FORMIN4 at the PM with meso-domains of PEN3 reveals a fine spatial segregation of destinations for actin-dependent immunity cargo. Together, our data suggest a model where FORMIN4 is a spatial feedback element in a multi-layered, temporally defined sequence of cytoskeletal response. This positional feedback makes a significant contribution to the distribution of actin filaments at the dynamic CWA boundary and to the outcomes of pre-invasion defense.

The cell wall of Arabidopsis thaliana influences actin network dynamics.

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility.

The copper spoil heap Knappenberg, Austria, as a model for metal habitats - Vegetation, substrate and contamination.

Historic mining in the Eastern Alps has left us with a legacy of numerous spoil heaps hosting specific, metal tolerant vegetation. Such habitats are characterized by elevated concentrations of toxic elements but also by high irradiation, a poorly developed substrate or extreme pH of the soil. This study investigates the distribution of vascular plants, mosses and lichens on a copper spoil heap on the ore bearing Knappenberg formed by Prebichl Layers and Werfener Schist in Lower Austria. It serves as a model for discriminating between various ecological traits and their effects on vegetation. Five distinct clusters were distinguished: (1) The bare, metal rich Central Spoil Heap was only colonised by highly resistant specialists. (2) The Northern and (3) Southern Peripheries contained less copper; the contrasting vegetation was best explained by the different microclimate. (4) A forest over acidic bedrock hosted a vegetation overlapping with the periphery of the spoil heap. (5) A forest over calcareous bedrock was similar to the spoil heap with regard to pH and humus content but hosted a vegetation differing strongly to all other habitats. Among the multiple toxic elements at the spoil heap, only Cu seems to exert a crucial influence on the vegetation pattern. Besides metal concentrations, irradiation, humidity, humus, pH and grain size distribution are important for the establishment of a metal tolerant vegetation. The difference between the species poor Northern and the diverse Southern Periphery can be explained by the microclimate rather than by the substrate. All plant species penetrating from the forest into the periphery of the spoil heap originate from the acidic but not from the calcareous bedrock.

Limited accumulation of copper in heavy metal adapted mosses.

Copper is an essential micronutrient but has toxic effects at high concentrations. Bryophytes are remarkably tolerant to elevated levels of copper but we wondered if this tolerance might be species dependent. Therefore, in three moss species, Physcomitrella patens, Mielichhoferia elongata and Pohlia drummondii, the accumulation of copper was compared with semiquantitative SEM-EDX analyses after six weeks of cultivation on copper containing media. We investigated the role of the copper-linked anion and applied copper as CuCl2, CuSO4 and CuEDTA, respectively. Line scans along the growth axis of moss gametophores allowed for a detailed analysis of copper detection from the base towards the tip. Mosses originating from metal-containing habitats (i.e. M. elongata and P. drummondii) revealed a lower accumulation of copper when compared to the non-adapted P. patens. CuEDTA had a shielding effect in all three species and copper levels differed greatly from CuCl2 or CuSO4. The detection of reactive oxygen species (ROS), H2O2 and O2(-), was further used to indicate stress levels in the gametophore stems. ROS staining was increased along the whole stem and the tip in the non-adapted species P. patens whereas the tolerant species M. elongata and P. drummondii generally showed less staining located mainly at the base of the stem. We discuss the relation between metal accumulation and ROS production using indicator dyes in the three moss species. As moss gametophores are very delicate structures, ROS staining provide an excellent alternative to spectrophotometric analyses to estimate stress levels.

Biochar decelerates soil organic nitrogen cycling but stimulates soil nitrification in a temperate arable field trial.

Biochar production and subsequent soil incorporation could provide carbon farming solutions to global climate change and escalating food demand. There is evidence that biochar amendment causes fundamental changes in soil nutrient cycles, often resulting in marked increases in crop production, particularly in acidic and in infertile soils with low soil organic matter contents, although comparable outcomes in temperate soils are variable. We offer insight into the mechanisms underlying these findings by focusing attention on the soil nitrogen (N) cycle, specifically on hitherto unmeasured processes of organic N cycling in arable soils. We here investigated the impacts of biochar addition on soil organic and inorganic N pools and on gross transformation rates of both pools in a biochar field trial on arable land (Chernozem) in Traismauer, Lower Austria. We found that biochar increased total soil organic carbon but decreased the extractable organic C pool and soil nitrate. While gross rates of organic N transformation processes were reduced by 50-80%, gross N mineralization of organic N was not affected. In contrast, biochar promoted soil ammonia-oxidizer populations (bacterial and archaeal nitrifiers) and accelerated gross nitrification rates more than two-fold. Our findings indicate a de-coupling of the soil organic and inorganic N cycles, with a build-up of organic N, and deceleration of inorganic N release from this pool. The results therefore suggest that addition of inorganic fertilizer-N in combination with biochar could compensate for the reduction in organic N mineralization, with plants and microbes drawing on fertilizer-N for growth, in turn fuelling the belowground build-up of organic N. We conclude that combined addition of biochar with fertilizer-N may increase soil organic N in turn enhancing soil carbon sequestration and thereby could play a fundamental role in future soil management strategies.

Plasmolysis: Loss of Turgor and Beyond.

Plasmolysis is a typical response of plant cells exposed to hyperosmotic stress. The loss of turgor causes the violent detachment of the living protoplast from the cell wall. The plasmolytic process is mainly driven by the vacuole. Plasmolysis is reversible (deplasmolysis) and characteristic to living plant cells. Obviously, dramatic structural changes are required to fulfill a plasmolytic cycle. In the present paper, the fate of cortical microtubules and actin microfilaments is documented throughout a plasmolytic cycle in living cells of green fluorescent protein (GFP) tagged Arabidopsis lines. While the microtubules became wavy and highly bundled during plasmolysis, cortical filamentous actin remained in close vicinity to the plasma membrane lining the sites of concave plasmolysis and adjusting readily to the diminished size of the protoplast. During deplasmolysis, cortical microtubule re-organization progressed slowly and required up to 24 h to complete the restoration of the original pre-plasmolytic pattern. Actin microfilaments, again, recovered faster and organelle movement remained intact throughout the whole process. In summary, the hydrostatic skeleton resulting from the osmotic state of the plant vacuole "overrules" the stabilization by cortical cytoskeletal elements.

Iron deprivation-induced reactive oxygen species generation leads to non-autolytic PCD in Brassica napus leaves.

Using iron-deprived (-Fe) chlorotic as well as green iron-deficient (5 µM Fe) and iron-sufficient supplied (50 µM Fe) leaves of young hydroponically reared Brassica napus plants, we explored iron deficiency effects on triggering programmed cell death (PCD) phenomena. Iron deficiency increased superoxide anion but decreased hydroxyl radical (•OH) formation (TBARS levels). Impaired photosystem II efficiency led to hydrogen peroxide accumulation in chloroplasts; NADPH oxidase activity, however, remained on the same level in all treatments. Non-autolytic PCD was observed especially in the chlorotic leaf of iron-deprived plants, to a lesser extent in iron-deficient plants. It correlated with higher DNAse-, alkaline protease- and caspase-3-like activities, DNA fragmentation and chromatin condensation, hydrogen peroxide accumulation and higher superoxide dismutase activity. A significant decrease in catalase activity together with rising levels of dehydroascorbic acid indicated a strong disturbance of the redox homeostasis, which, however, was not caused by •OH formation in concordance with the fact that iron is required to catalyse the Fenton reaction leading to •OH generation. This study documents the chain of events that contributes to the development of non-autolytic PCD in advanced stages of iron deficiency in B. napus leaves.

Comparing copper resistance in two bryophytes: Mielichhoferia elongata Hornsch. versus Physcomitrella patens Hedw.

The bryophyte Mielichhoferia elongata is known to occur on copper-rich substrate, but the exact resistance level remained to be determined by in vitro experiments. Here, we tested its copper tolerance in graded copper solutions and compared the results to the moss Physcomitrella patens that is not known to inhabit heavy metal sites. Our results confirm the survival of M. elongata in classical resistance experiments of up to 10 mM Cu-ethylenediaminetetraacetic acid (EDTA) solution. Interestingly, P. patens is equally resistant. Cultured on copper-enriched agar plates for over 5 weeks, P. patens survived even higher copper levels of up to 100 mM Cu-EDTA and an increment of growth was detected on all concentrations tested. Obviously, P. patens is able to withstand harmfully high levels of copper in both solution and substrate. In this short communication, we give a detailed description of the growth rates and discuss the results in comparison to other moss species and heavy metals.